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WO2008154628A2 - Procédé pour la stérilisation d'un tissu mou acellulaire sous vide - Google Patents

Procédé pour la stérilisation d'un tissu mou acellulaire sous vide Download PDF

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Publication number
WO2008154628A2
WO2008154628A2 PCT/US2008/066697 US2008066697W WO2008154628A2 WO 2008154628 A2 WO2008154628 A2 WO 2008154628A2 US 2008066697 W US2008066697 W US 2008066697W WO 2008154628 A2 WO2008154628 A2 WO 2008154628A2
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WO
WIPO (PCT)
Prior art keywords
skin
soft tissue
tissue
human
dermis
Prior art date
Application number
PCT/US2008/066697
Other languages
English (en)
Other versions
WO2008154628A3 (fr
Inventor
Manh-Dan Ngo
Jeffrey Stuart Cartmell
Arthur A. Gertzman
Original Assignee
Musculoskeletal Transplant Foundation
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Application filed by Musculoskeletal Transplant Foundation filed Critical Musculoskeletal Transplant Foundation
Priority to US12/664,296 priority Critical patent/US20100296969A1/en
Publication of WO2008154628A2 publication Critical patent/WO2008154628A2/fr
Publication of WO2008154628A3 publication Critical patent/WO2008154628A3/fr

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Classifications

    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/50—Materials characterised by their function or physical properties, e.g. injectable or lubricating compositions, shape-memory materials, surface modified materials
    • A61L27/60—Materials for use in artificial skin
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L2/00—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor
    • A61L2/0005—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts
    • A61L2/0082—Methods or apparatus for disinfecting or sterilising materials or objects other than foodstuffs or contact lenses; Accessories therefor for pharmaceuticals, biologicals or living parts using chemical substances
    • A61L2/0094—Gaseous substances
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3604—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
    • A61L27/362—Skin, e.g. dermal papillae
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3687—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by the use of chemical agents in the treatment, e.g. specific enzymes, detergents, capping agents, crosslinkers, anticalcification agents
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61L—METHODS OR APPARATUS FOR STERILISING MATERIALS OR OBJECTS IN GENERAL; DISINFECTION, STERILISATION OR DEODORISATION OF AIR; CHEMICAL ASPECTS OF BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES; MATERIALS FOR BANDAGES, DRESSINGS, ABSORBENT PADS OR SURGICAL ARTICLES
    • A61L27/00—Materials for grafts or prostheses or for coating grafts or prostheses
    • A61L27/36—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix
    • A61L27/3683—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment
    • A61L27/3691—Materials for grafts or prostheses or for coating grafts or prostheses containing ingredients of undetermined constitution or reaction products thereof, e.g. transplant tissue, natural bone, extracellular matrix subjected to a specific treatment prior to implantation, e.g. decellularising, demineralising, grinding, cellular disruption/non-collagenous protein removal, anti-calcification, crosslinking, supercritical fluid extraction, enzyme treatment characterised by physical conditions of the treatment, e.g. applying a compressive force to the composition, pressure cycles, ultrasonic/sonication or microwave treatment, lyophilisation

Definitions

  • the present invention is generally directed toward methods of treatment of soft tissue including decellularizing and sterilization of the soft tissue by placing the same under vacuum and treating the soft tissue with an oxide or allotropic form of oxygen for a period of time for implantation into another human being.
  • Tissue transplantation is another way of restoring function by replacing, regenerating, repairing, rebuilding or protecting the damaged tissue.
  • Immunosuppressive drugs such as cyclosporin and FK506 are usually given to the patient to prevent rejection. These immunosuppressive drugs however, have a narrow therapeutic window between adequate immunosuppression and toxicity. Prolonged immunosuppression can weaken the immune system, which can lead to a threat of infection.
  • the present invention is directed toward a process for use in the preparation of acellular, i.e. (essentially lacking in living cells and/or non-living cells,) soft-tissue implants derived from tissue products taken from mammals and in particular the skin of human donors.
  • acellular i.e. (essentially lacking in living cells and/or non-living cells,) soft-tissue implants derived from tissue products taken from mammals and in particular the skin of human donors.
  • the decellularized grafts produced are significantly improved in long-term durability and function when used in clinical applications.
  • U.S. Patent Number 4,776,853 issued October 11, 1988 is directed toward a process for preparing biological material for implant in a mammal's cardiovascular system, respiratory system or soft tissue.
  • the process comprises: (1) isolating a desired tissue sample of the biological material from a donor; (2) extracting the tissue sample with an hypotonic buffer solution at a mild alkaline pH, the buffer solution including active amounts of proteolytic inhibitors and antibiotics; (3) extracting the tissue sample with a buffered solution having a high concentration of salt, the solution being at a mild alkaline pH and including a non-ionic detergent with protease inhibitors and antibiotics; (4) subjecting the tissue sample to enzymatic digestion in a buffered saline solution, the enzymes consisting of purified protease-free dioxyribonuclease and ribonuclease; (5) extracting the tissue sample with an anionic detergent at a mild alkaline pH; and (6) storing the tissue sample in physiologic buffered
  • U.S. Patent Number 6,734,018 issued May 11, 2004 which is directed toward a process for preparing an acellular soft tissue graft for implantation into a mammalian system.
  • the process extracts a soft tissue sample with an extracting solution including one or more nonionic detergents and one or more endonucleases, to produce extracted tissue and treats the extracted tissue with a treating solution including one or more anionic detergents, to produce a treated tissue.
  • the treated tissue is washed with a decontaminating solution to produce the acellular soft tissue graft; and the acellular soft tissue graft is then stored in a storage solution comprising one or more decontaminating agents.
  • the soft tissue process of the '018 patent includes the steps of: isolating from a suitable donor a desired tissue sample of the biological material; extracting the tissue with mildly alkaline hypotonic buffered solution of an endonuclease such as Benzonase RTM and a nonionic detergent formulation such as Allowash SolutionTM, optionally treating the tissue with a hypertonic buffered salt solution; extracting and treating the tissue with a mildly alkaline hypotonic buffered solution of sodium dodecylsulfate, optionally with 0.1 to 0.5 M sodium chloride rendering the solution hypertonic; washing the tissue with ultrapure water followed by a water solution of chloride dioxide; and storage in a sealed container in isotonic saline, chloride dioxide or 70% isopropanol.
  • the present invention is a process for preparing soft tissue for implant in a human and removes cellular components from tissue taken from a mammal while sterilizing the tissue under vacuum while treating the same with an oxide or an allotropic form of oxygen.
  • the process comprises the following steps:
  • Figure 1 is a schematic flow chart showing the soft tissue decellularization and sterilization process.
  • the present invention is directed towards the preparation of soft tissue from a mammal, preferably from a human which is processed, decellularized and sterilized.
  • the preferred form of soft tissue is skin although other forms of soft tissue can be treated.
  • the soft tissue which is envisioned as being processed is full thickness skin which includes the epidermis, dermis and subcutaneous layers.
  • the epidermis is the outer most layer of the skin and dermis is the layer of skin lying immediately under the epidermis and the term skin may refer to either epidermis, dermis or subcutaneous layers or all of the same, depending on the usage.
  • the skin which has been previously obtained from a donor who is deceased or living is shipped from the donor site in a container which may contain antibiotics, alcohol or mixtures of same, mixed with a decellularizing solution such as Sodium Chloride and is then frozen. This minimizes or prevents contamination of the tissue and begins the epidermal separation from the dermal skin layer.
  • the frozen skin is then taken from the freezer and thawed in a basin filled with sterile purified water.
  • tissue Prior to processing, tissue is inspected for damage (holes or tears) and distinctive features (moles, warts, tattoos) which are removed using a scalpel.
  • the tissue is inspected for hair and the same is removed using anyone of a number of techniques including chemical removal using compositions such as (1) water, mineral oil, calcium thioglycolate, calcium hydroxide, ceteareth-20, sodium hydroxide, camellia oleifera extract, sunflower seed oil, fragrance, chromium hydroxide green and alkaline soap and physical removal such as (2) hot wax, hair inhibition, non-heating type laser hair removal in ultra short pulse (USP) range and microdermabrasion.
  • a visual inspection is performed to ensure the skin tissue has uniform thickness. Thickness is recorded using a thickness gauge.
  • To identify the orientation (dermal or epidermal side) of tissue such as skin the skin is positioned such that the epidermis faces the processor and an incision is cut into the upper left comer of each piece of tissue to indicate the epidermal side.
  • the tissue form Prior to processing, the tissue form is inspected for visual defects and then trimmed for further processing.
  • the epidermal layer is removed and the dermis is decellularized using Sodium Chloride (NaCl) solution at a concentration of 0.1 - 1OM, preferably about IM with a pH ranging from 5.0 - 9.0, preferably 6.8 - 7.2, and is agitated at a speed of 65 rpm on an orbital shaker for 1-96 hours, preferably 12 hours to a maximum of 48 hours.
  • NaCl Sodium Chloride
  • the container holding the skin is checked to ascertain if the epidermal layers have been sloughed off. If not, the container is checked every 2 hours.
  • the dermis is then removed and placed on a cutting board with the epidermal side up and any remaining epidermal layers are picked off and discarded as well as any remaining hairs.
  • the remaining dermis pieces are replaced in the tissue flasks, filled with sterile water and agitated on the orbital shaker for 15 minutes. The sterile water is refreshed and the rinse procedure is repeated one more time for a total of two rinses.
  • the dermis pieces are trimmed into shaped pieces, preferably rectangular, by removing all of the rough edges of each piece with a scalpel.
  • the trimmed dermis pieces are then immersed in 0.1% Triton X-IOO solution having a concentration of 0.01 - 10.0%, preferably about 0.1% with a pH ranging from 4.5 - 8.5, preferably 6.2 - 7.0 and agitated on the orbital shaker for 1 - 96 hours, preferably 24 hours to 48 hours.
  • the dermis is then placed in tissue flasks filled with sterile water, and agitated on the orbital shaker at 65 rpm for 15 minutes.
  • the sterile water is refreshed and the rinse procedure is repeated a minimum of 5 more times for a total of 6 water rinses.
  • a residual detergent test is performed on the rinsate after the 6 th water rinse to ensure the detergent has been adequately removed.
  • the treated soft tissue in the nature of acellular dermis is sterilized under vacuum with an allotropic form of oxygen such as Ozone (TSO 3 ) at a temperature ranging from 10° C - 50° C for a period ranging from about 15 minutes to about 120 minutes to achieve a dosage concentration of about 5% to about 15% or by using various oxides taken from a group of Vapor H 2 O 2 (VHP) at a temperature ranging from 20° C - 50° C for a period ranging from about 30 minutes to about 180 minutes; Plasma H 2 O 2 at a temperature ranging from 30° C - 60° C for a period ranging from 30 - 180 minutes; Ethylene Oxide Gas at a temperature ranging from about 35° C to about 70°C for period ranging from about 12 hours to about 20 hours to achieve a dosage concentration of 100 - 1000 ppm or by using Sodium Hydroxide (NaOH) at a temperature of 10° C - 30° C for a period ranging from about 0.5 hours to about
  • the tissue which has been previously obtained from a donor is shipped from the donor site in a container having a sterilization solution mixed with a decellularizing solution such as sodium chloride and then frozen.
  • the donor tissue is then thawed and then rinsed to maintain moisture.
  • the thawed tissue is processed by removing hair and is then decellularized using IM NaCl and 0.1% of Triton X-IOO.
  • one or more of the following protease inhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (serine proteases) (25-100 ⁇ m, Aprotinin (broad spectrum, serine proteases) (7.5-30 ⁇ m), Protease Inhibitor E-64 (cysteine proteases) (0.05-.0.20), Leupeptin, Hemisulfate (cysteine proteases) (0.05-.0.20 ⁇ m), EDTA, Disodium (0.025-.0.10 ⁇ m), and trypsin-like proteases, Pepstatin A (Aspartic Proteases), Marmistat (MMP2).
  • the tissue is processed and decellularized and is
  • Each skin piece is checked for hairs and the hairs are removed chemically by application of chemical compositions such as water, mineral oil, calcium thioglycolate, calcium hydroxide, ceteareth-20, sodium hydroxide, camellia oleifera extract, sunflower seed oil, fragrance, chromium hydroxide green after which the skin is rinsed with water.
  • the skin is positioned with the dermis side up (epidermis down) on the cutting board and rectangular skin pieces are cut by removing the rough edges of each piece with one or more uninterrupted cuts using a scalpel and ruler. An incision is cut into the left hand comer of each piece of skin indicating the epidermal side of the skin. A visual inspection is performed to make sure the tissue has a uniform thickness throughout the piece and regions with a visibly low or nonuniform thickness are removed. A thickness measurement is then performed using a thickness gauge.
  • the skin is decellularized in a sterile tissue culture bottle filled with IL of IM NaCl.
  • the bottle is sealed in a self-seal pouch and the bottle is placed on its flat side on the shaker with a set speed of 65 rpm for a period of 12 - 48 hours.
  • the bottle(s) is checked after the first 12 hours to see if the epidermal layers have sloughed off. After the first 12 hour check, the bottle is checked every 2 hours until all epidermal layers have been sloughed.
  • the bottles are removed from the shaker and the NaCl is emptied from the bottle(s).
  • the skin is removed from the bottle and placed on the cutting board with the epidermal side up.
  • the epidermal layers are peeled off with forceps and discarded leaving only the dermal layer (dermis).
  • the bottles are rinsed with sterile water and the peeled skin pieces (dermis) are placed back into the bottle.
  • the bottles are then filled with enough sterile water to submerge the tissue while the bottle is lying flat and the bottle is placed on the shaker which has a preset speed of 65 rpm.
  • the shaker is set to run for 15 minutes. After running 15 minutes, the bottle(s) are removed and the water is changed with clean sterile water. This rinse is repeated one more time for a total of two times.
  • the bottle(s) are removed from the shaker, emptied and filled with IL of 0.1% Triton X-IOO.
  • the bottle containing the dermis is seated in a self-seal pouch and placed on the shaker set to the speed to 65 rpm's and allowed to shake for 24 to 48 hours.
  • the shaker is stopped after 24 hours or a later time period, the dermis is removed from the bottles and place submerged in a container with sterile water to rinse off the Triton X-100.
  • the tissue is again rinsed with a sterile water for 15 minutes at 65 rpm's for irrigation to rinse off the Triton X-100.
  • the rinse is repeated 5 more times for a total of 6 times. After rinsing a residual detergent test is performed to make sure that the detergent has been removed from the tissue so that less than lppm is found on the tissue.
  • the tissue may be lyophilized or is immersed in 70% ethanol and 30% water and packaged for storage in sterile foil.
  • the skin is laid flat on screens and placed in a double. Tyvek® pouch.
  • the tissue is placed in a freezer at -70° on the lyophilization staging shelf to prevent the tissue from becoming wrinkled or deformed until the lyophilizer is available.
  • the dermis tissue is cut to size and may be perforated with the perforations 10 spaced 2-3mm apart with each perforation preferably having a diameter of about 1.2mm.
  • the dermis is then sterilized under vacuum with Ozone (TSO3) at a preferred temperature ranging from 30° C - 35° C for a preferred period of about 1 hour to achieve a concentration of about 6% to about 12%. After treatment the dermis is sterile.
  • Ozone Ozone
  • Example 2 Sterilization of Dermis under Vacuum with Vapor H2O2.
  • the tissue which has been previously obtained from a donor is shipped from the donor site in a container having a sterilization solution mixed with a decellularizing solution such as sodium chloride and then frozen.
  • the donor tissue is then thawed and then rinsed to maintain moisture.
  • the thawed tissue is processed by removing hair and is then decellularized using IM NaCl and 0.1% of Triton X-100.
  • one or more of the following protease inhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (serine proteases) (25-100 ⁇ m, Aprotinin (broad spectrum, serine proteases) (7.5-30 ⁇ m), Protease Inhibitor E-64 (cysteine proteases) (0.05-.0.20 ⁇ m), Leupeptin, Hemisulfate (cysteine proteases) (0.05-.0.20 ⁇ m), EDTA, Disodium (0.025-.0.10 ⁇ m), and trypsin-like proteases, Pepstatin A (Aspartic Proteases), Marmistat (MMP2).
  • the tissue is processed and decellularized and is
  • Each skin piece is checked for hairs and the hairs are removed chemically by application of chemical compositions such as water, mineral oil, calcium thioglycolate, calcium hydroxide, ceteareth-20, sodium hydroxide, camellia oleifera extract, sunflower seed oil, fragrance, chromium hydroxide green after which the skin is rinsed with water.
  • the skin is positioned with the dermis side up (epidermis down) on the cutting board and rectangular skin pieces are cut by removing the rough edges of each piece with one or more uninterrupted cuts using a scalpel and ruler. An incision is cut into the left hand corner of each piece of skin indicating the epidermal side of the skin. A visual inspection is performed to make sure the tissue has a uniform thickness throughout the piece and regions with a visibly low or nonuniform thickness are removed. A thickness measurement is then performed using a thickness gauge.
  • the skin is decellularized in a sterile tissue culture bottle filled with IL of IM NaCl.
  • the bottle is sealed in a self-seal pouch and the bottle is placed on its flat side on the shaker with a set speed of 65 rpm for a period of 12 - 48 hours.
  • the bottle(s) is checked after the first 12 hours to see if the epidermal layers have sloughed off. After the first 12 hour check, the bottle is checked every 2 hours until all epidermal layers have been sloughed.
  • the bottles are removed from the shaker and the NaCl is emptied from the bottle(s).
  • the skin is removed from the bottle and placed on the cutting board with the epidermal side up.
  • the epidermal layers are peeled off with forceps and discarded leaving only the dermal layer (dermis).
  • the bottles are rinsed with sterile water and the peeled skin pieces (dermis) are placed back into the bottle.
  • the bottles are then filled with enough sterile water to submerge the tissue while the bottle is lying flat and the bottle is placed on the shaker which has a preset speed of 65 rpm.
  • the shaker is set to run for 15 minutes. After running 15 minutes, the bottle(s) are removed and the water is changed with clean sterile water. This rinse is repeated one more time for a total of two times.
  • the bottle(s) are removed from the shaker, emptied and filled with IL of 0.1% Triton X-IOO.
  • the bottle containing the dermis is seated in a self-seal pouch and placed on the shaker set to the speed to 65 rpm's and allowed to shake for 24 to 48 hours.
  • the shaker is stopped after 24 hours or a later time period, the dermis is removed from the bottles and place submerged in a container with sterile water to rinse off the Triton X-100.
  • the tissue is again rinsed with a sterile water for 15 minutes at 65 rpm's for irrigation to rinse off the Triton X-100.
  • the rinse is repeated 5 more times for a total of 6 times. After rinsing a residual detergent test is performed to make sure that the detergent has been removed from the tissue so that less than lppm is found on the tissue.
  • the tissue may be lyophilized or is immersed in 70% ethanol and 30% water and packaged for storage in sterile foil.
  • the skin is laid flat on screens and placed in a double Tyvek® pouch.
  • the tissue is placed in a freezer at -70° on the lyophilization staging shelf to prevent the tissue from becoming wrinkled or deformed until the lyophilizer is available.
  • the dermis tissue Upon removal from the lyophilization, the dermis tissue is cut to size and may be perforated with the perforations 10 spaced 2-3 mm apart with each perforation preferably having a diameter of about 1.2mm.
  • the dermis is then sterilized under vacuum with Vapor H 2 O 2 (VHP) at a preferred temperature ranging from about 30°C to about 40 0 C for a preferred period ranging from about 0.5 hours to about 3 hours. After treatment the dermis is sterile.
  • VHP Vapor H 2 O 2
  • the tissue which has been previously obtained from a donor is shipped from the donor site in a container having a sterilization solution mixed with a decellularizing solution such as sodium chloride and then frozen.
  • the donor tissue is then thawed and then rinsed to maintain moisture.
  • the thawed tissue is processed by removing hair and is then decellularized using IM NaCl and 0.1% of Triton X-IOO.
  • one or more of the following protease inhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (serine proteases) (25-100 ⁇ m, Aprotinin (broad spectrum, serine proteases) (7.5-30 ⁇ m), Protease Inhibitor E-64 (cysteine proteases) (0.05-.0.20 ⁇ m), Leupeptin, Hemisulfate (cysteine proteases) (0.05-.0.20 ⁇ m), EDTA, Disodium (0.025-.0.10 ⁇ m), and trypsin-like proteases, Pepstatin A (Aspartic Proteases), Marmistat (MMP2).
  • the tissue is processed and
  • Each skin piece is checked for hairs and the hairs are removed chemically by application of chemical compositions such as water, mineral oil, calcium thioglycolate, calcium hydroxide, ceteareth-20, sodium hydroxide, camellia oleifera extract, sunflower seed oil, fragrance, chromium hydroxide green after which the skin is rinsed with water.
  • the skin is positioned with the dermis side up (epidermis down) on the cutting board and rectangular skin pieces are cut by removing the rough edges of each piece with one or more uninterrupted cuts using a scalpel and ruler. An incision is cut into the left hand comer of each piece of skin indicating the epidermal side of the skin. A visual inspection is performed to make sure the tissue has a uniform thickness throughout the piece and regions with a visibly low or nonuniform thickness are removed. A thickness measurement is then performed using a thickness gauge.
  • the skin is decellularized in a sterile tissue culture bottle filled with IL of IM NaCl.
  • the bottle is sealed in a self-seal pouch and the bottle is placed on its flat side on the shaker with a set speed of 65 rpm for a period of 12 - 48 hours.
  • the bottle(s) is checked after the first 12 hours to see if the epidermal layers have sloughed off. After the first 12 hour check, the bottle is checked every 2 hours until all epidermal layers have been sloughed.
  • the bottles are removed from the shaker and the NaCl is emptied from the bottle(s).
  • the skin is removed from the bottle and placed on the cutting board with the epidermal side up.
  • the epidermal layers are peeled off with forceps and discarded leaving only the dermal layer (dermis).
  • the bottles are rinsed with sterile water and the peeled skin pieces (dermis) are placed back into the bottle.
  • the bottles are then filled with enough sterile water to submerge the tissue while the bottle is lying flat and the bottle is placed on the shaker which has a preset speed of 65 rpm.
  • the shaker is set to run for 15 minutes. After running 15 minutes, the bottle(s) are removed and the water is changed with clean sterile water. This rinse is repeated one more time for a total of two times.
  • the bottle(s) are removed from the shaker, emptied and filled with IL of 0.1% Triton X-IOO.
  • the bottle containing the dermis is seated in a self-seal pouch and placed on the shaker set to the speed to 65 rpm's and allowed to shake for 24 to 48 hours.
  • the shaker is stopped after 24 hours or a later time period, the dermis is removed from the bottles and place submerged in a container with sterile water to rinse off the Triton X-100.
  • the tissue is again rinsed with a sterile water for 15 minutes at 65 rpm's for irrigation to rinse off the Triton X-100.
  • the rinse is repeated 5 more times for a total of 6 times. After rinsing a residual detergent test is performed to make sure that the detergent has been removed from the tissue so that less than lppm is found on the tissue.
  • the tissue may be lyophilized or is immersed in 70% ethanol and 30% water and packaged for storage in sterile foil.
  • the skin is laid flat on screens and placed in a double Tyvek® pouch.
  • the tissue is placed in a freezer at -70° on the lyophilization staging shelf to prevent the tissue from becoming wrinkled or deformed until the lyophilizer is available.
  • the dermis tissue is cut to size and may be perforated with the perforations 10 spaced 2-3mm apart with each perforation preferably having a diameter of about 1.2mm.
  • the dermis is then treated under vacuum with Plasma H 2 O 2 at a preferred temperature ranging from about 45 0 C to about 50 0 C for a preferred period ranging from about 55 minutes to about 70 minutes. After treatment the dermis is sterile.
  • the tissue which has been previously obtained from a donor is shipped from the donor site in a container having a sterilization solution mixed with a decellularizing solution such as sodium chloride and then frozen.
  • the donor tissue is then thawed and then rinsed to maintain moisture.
  • the thawed tissue is processed by removing hair and is then decellularized using IM NaCl and 0.1% of Triton X-100.
  • one or more of the following protease inhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (serine proteases) (25-100 ⁇ m Aprotinin (broad spectrum, serine proteases) (7.5-3 ⁇ m), Protease Inhibitor E-64 (cysteine proteases) (0.05-.0.20 ⁇ m), Leupeptin, Hemisulfate (cysteine proteases) (0.05-.0.20 ⁇ m), EDTA, Disodium (0.025-.0.1 ⁇ m), and trypsin-like proteases, Pepstatin A (Aspartic Proteases), Marmistat (MMP2).
  • the tissue is processed and decellularized
  • Each skin piece is checked for hairs and the hairs are removed chemically by application of chemical compositions such as water, mineral oil, calcium thioglycolate, calcium hydroxide, ceteareth-20, sodium hydroxide, camellia oleifera extract, sunflower seed oil, fragrance, chromium hydroxide green after which the skin is rinsed with water.
  • the skin is positioned with the dermis side up (epidermis down) on the cutting board and rectangular skin pieces are cut by removing the rough edges of each piece with one or more uninterrupted cuts using a scalpel and ruler. An incision is cut into the left hand comer of each piece of skin indicating the epidermal side of the skin. A visual inspection is performed to make sure the tissue has a uniform thickness throughout the piece and regions with a visibly low or nonuniform thickness are removed. A thickness measurement is then performed using a thickness gauge.
  • the skin is decellularized in a sterile tissue culture bottle filled with IL of IM NaCl.
  • the bottle is sealed in a self-seal pouch and the bottle is placed on its flat side on the shaker with a set speed of 65 rpm for a period of 12 - 48 hours.
  • the bottle(s) is checked after the first 12 hours to see if the epidermal layers have sloughed off. After the first 12 hour check, the bottle is checked every 2 hours until all epidermal layers have been sloughed.
  • the bottles are removed from the shaker and the NaCl is emptied from the bottle(s).
  • the skin is removed from the bottle and placed on the cutting board with the epidermal side up.
  • the epidermal layers are peeled off with forceps and discarded leaving only the dermal layer (dermis).
  • the bottles are rinsed with sterile water and the peeled skin pieces (dermis) are placed back into the bottle.
  • the bottles are then filled with enough sterile water to submerge the tissue while the bottle is lying flat and the bottle is placed on the shaker which has a preset speed of 65 rpm.
  • the shaker is set to run for 15 minutes. After running 15 minutes, the bottle(s) are removed and the water is changed with clean sterile water. This rinse is repeated one more time for a total of two times.
  • the bottle(s) are removed from the shaker, emptied and filled with IL of 0.1% Triton X-IOO.
  • the bottle containing the dermis is seated in a self-seal pouch and placed on the shaker set to the speed to 65 rpm's and allowed to shake for 24 to 48 hours.
  • the shaker is stopped after 24 hours or a later time period, the dermis is removed from the bottles and place submerged in a container with sterile water to rinse off the Triton X-100.
  • the tissue is again rinsed with a sterile water for 15 minutes at 65 rpm's for irrigation to rinse off the Triton X-100.
  • the rinse is repeated 5 more times for a total of 6 times. After rinsing a residual detergent test is performed to make sure that the detergent has been removed from the tissue so that less than lppm is found on the tissue.
  • the tissue may be lyophilized or is immersed in 70% ethanol and 30% water and packaged for storage in sterile foil.
  • the skin is laid flat on screens and placed in a double Tyvek® pouch.
  • the tissue is placed in a freezer at -70° on the lyophilization staging shelf to prevent the tissue from becoming wrinkled or deformed until the lyophilizer is available.
  • the dermis tissue Upon removal from the lyophilization, the dermis tissue is cut to size and may be perforated with the perforations 10 spaced 2-3mm apart with each perforation preferably having a diameter of about 1.2mm.
  • the dermis is then treated under vacuum with Ethylene Oxide Gas at a preferred temperature ranging from about 50 0 C to about 60 0 C for a preferred period ranging from about 16 to about 18 hours to achieve a dose or concentration of about 100 -lOOOppm. After treatment the dermis is sterile.
  • the tissue which has been previously obtained from a donor is shipped from the donor site in a container sterilization solution mixed with a decellularizing solution such as sodium chloride and then frozen.
  • the donor tissue is then thawed and then rinsed to maintain moisture.
  • the thawed tissue is processed by removing hair and is then decellularized using IM NaCl and 0.1% of Triton X-IOO.
  • protease inhibitors may be added; Aminoethylbenzenesulfonyl fluoride HCL (serine proteases) (25-100 ⁇ m, Aprotinin (broad spectrum, serine proteases) (7.5-30 ⁇ m), Protease Inhibitor E-64 (cysteine proteases) (0.05-.0.20 ⁇ m), Leupeptin, Hemisulfate (cysteine proteases) (0.05-.0.20 ⁇ m), EDTA, Disodium (0.025-.0.10 ⁇ m), and trypsin-like proteases, Pepstatin A (Aspartic Proteases), Marmistat (MMP2). The tissue is processed and decellularized and is inspected for visual defects and trimmed.
  • Aminoethylbenzenesulfonyl fluoride HCL serine proteases
  • Protease Inhibitor E-64 cysteine proteases
  • Leupeptin 0.05-.
  • Each skin piece is checked for hairs and the hairs are removed chemically by application of chemical compositions such as water, mineral oil, calcium thioglycolate, calcium hydroxide, ceteareth-20, sodium hydroxide, camellia oleifera extract, sunflower seed oil, fragrance, chromium hydroxide green after which the skin is rinsed with water.
  • the skin is positioned with the dermis side up (epidermis down) on the cutting board and rectangular skin pieces are cut by removing the rough edges of each piece with one or more uninterrupted cuts using a scalpel and ruler. An incision is cut into the left hand comer of each piece of skin indicating the epidermal side of the skin.
  • a visual inspection is performed to make sure the tissue has a uniform thickness throughout the piece and regions with a visibly low or nonuniform thickness are removed.
  • a thickness measurement is then performed using a thickness gauge.
  • the skin is decellularized in a sterile tissue culture bottle filled with IL of IM NaCl.
  • the bottle is sealed in a self-seal pouch and the bottle is placed on its flat side on the shaker with a set speed of 65 rpm for a period of 12 - 48 hours.
  • the bottle(s) is checked after the first 12 hours to see if the epidermal layers have sloughed off. After the first 12 hour check, the bottle is checked every 2 hours until all epidermal layers have been sloughed.
  • the bottles are removed from the shaker and the NaCl is emptied from the bottle(s).
  • the skin is removed from the bottle and placed on the cutting board with the epidermal side up.
  • the epidermal layers are peeled off with forceps and discarded leaving only the dermal layer (dermis).
  • the bottles are rinsed with sterile water and the peeled skin pieces (dermis) are placed back into the bottle.
  • the bottles are then filled with enough sterile water to submerge the tissue while the bottle is lying flat and the bottle is placed on the shaker which has a preset speed of 65 rpm.
  • the shaker is set to run for 15 minutes. After running 15 minutes, the bottle(s) are removed and the water is changed with clean sterile water.
  • This rinse is repeated one more time for a total of two times.
  • the bottle(s) are removed from the shaker, emptied and filled with IL of 0.1% Triton X-IOO.
  • the bottle containing the dermis is seated in a self- seal pouch and placed on the shaker set to the speed to 65 rpm's and allowed to shake for 24 to 48 hours.
  • the shaker is stopped after 24 hours or a later time period, the dermis is removed from the bottles and place submerged in a container with sterile water to rinse off the Triton X-100.
  • the tissue is again rinsed with a sterile water for 15 minutes at 65 rpm's for irrigation to rinse off the Triton X-100.
  • the rinse is repeated 5 more times for a total of 6 times. After rinsing a residual detergent test is performed to make sure that the detergent has been removed from the tissue so that less than lppm is found on the tissue.
  • the tissue may be lyophilized or is immersed in 70% ethanol and 30% water and packaged for storage in sterile foil.
  • the skin is laid flat on screens and placed in a double Tyvek® pouch.
  • the tissue is placed in a freezer at -70° on the lyophilization staging shelf to prevent the tissue from becoming wrinkled or deformed until the lyophilizer is available.
  • the dermis tissue Upon removal from the lyophilization, the dermis tissue is cut to size and may be perforated with the perforations 10 spaced 2-3mm apart with each perforation preferably having a diameter of about 1.2mm.
  • the dermis is then treated under vacuum with Sodium Hydroxide (NaOH) at a preferred temperature ranging from 10 0 C to about 30 0 C for a preferred period ranging from about 0.5 to about 12 hours to achieve a concentration of about IM. After treatment the dermis is sterile.
  • NaOH Sodium Hydroxide

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  • Public Health (AREA)
  • Veterinary Medicine (AREA)
  • Molecular Biology (AREA)
  • Transplantation (AREA)
  • Oral & Maxillofacial Surgery (AREA)
  • Dermatology (AREA)
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  • Medicines That Contain Protein Lipid Enzymes And Other Medicines (AREA)

Abstract

La présente invention concerne un procédé destiné à la préparation de peau prélevée chez un donneur humain, à l'élimination de composants cellulaires et à la stérilisation de la peau décellularisée. Ce procédé comprend les étapes consistant (a) à décellulariser la peau, notamment à faire tremper la peau dans un détergent et à la rincer avec de l'eau stérile, (b) à stériliser la peau sous vide avec de l'ozone et/ou H2O2 sous forme vapeur et/ou H2O2 sous forme plasma et/ou du gaz d'oxyde d'éthylène et/ou de l'hydroxyde de sodium pendant une certaine péridoe de temps afin d'obtenir une concentration pour réaliser la stérilisation de la peau, et (c) à traiter le peau par le biais de sa coupe à une dimension spécifique.
PCT/US2008/066697 2007-06-12 2008-06-12 Procédé pour la stérilisation d'un tissu mou acellulaire sous vide WO2008154628A2 (fr)

Priority Applications (1)

Application Number Priority Date Filing Date Title
US12/664,296 US20100296969A1 (en) 2007-06-12 2008-06-12 Process for sterilizing acellular soft tissue under vacuum

Applications Claiming Priority (2)

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US92908507P 2007-06-12 2007-06-12
US60/929,085 2007-06-12

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WO2008154628A3 WO2008154628A3 (fr) 2009-02-12

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Cited By (3)

* Cited by examiner, † Cited by third party
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CN103357055A (zh) * 2013-08-02 2013-10-23 安徽汇仁堂中药饮片股份有限公司 一种中药材灭菌方法
US9566369B2 (en) 2010-02-26 2017-02-14 Decell Technologies Inc. Methods for tissue decellularization
US10307510B2 (en) 2013-11-04 2019-06-04 Lifecell Corporation Methods of removing alpha-galactose

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WO2011047127A1 (fr) 2009-10-15 2011-04-21 Minntech Corporation Système de désinfection de pièce par brumisation
EP2506884B1 (fr) 2009-12-03 2015-02-18 Minntech Corporation Récipient pour la décontamination d' un appareil médical avec de brouillard
CN102178981B (zh) * 2011-04-20 2013-08-28 北京市创伤骨科研究所 一种软骨修复支架材料的制备方法
US9017607B2 (en) 2011-05-27 2015-04-28 Medivators Inc. Decontamination system including environmental control using a decontaminating substance
CN104458376A (zh) * 2014-12-10 2015-03-25 贵州大学 一种软化昆虫外生殖器的方法
CN112587697B (zh) * 2020-12-15 2022-07-26 马东骏 消杀冷链物品中新冠病毒的装置及方法
KR102556908B1 (ko) * 2022-08-05 2023-07-20 주식회사 지씨에스 플라즈마 발생 장치

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Publication number Priority date Publication date Assignee Title
WO1998015297A1 (fr) * 1996-10-09 1998-04-16 Lifetech Corporation Procede de sterilisation de matieres biologiques
GB9825938D0 (en) * 1998-11-27 1999-01-20 Univ Sheffield Skin composites
US20030068815A1 (en) * 1999-02-11 2003-04-10 Stone Kevin R. Sterilized xenograft tissue
US6875018B2 (en) * 2001-03-28 2005-04-05 Curozone Ireland Limited Use of ozone for the treatment of root canals

Cited By (4)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US9566369B2 (en) 2010-02-26 2017-02-14 Decell Technologies Inc. Methods for tissue decellularization
CN103357055A (zh) * 2013-08-02 2013-10-23 安徽汇仁堂中药饮片股份有限公司 一种中药材灭菌方法
CN103357055B (zh) * 2013-08-02 2016-07-06 安徽汇仁堂中药饮片股份有限公司 一种中药材灭菌方法
US10307510B2 (en) 2013-11-04 2019-06-04 Lifecell Corporation Methods of removing alpha-galactose

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WO2008154628A3 (fr) 2009-02-12

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